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Characterization of Ca 2+ ATPase Residues Involved in Substrate and Cation Binding
Author(s) -
INESI GIUSEPPE,
MA HAILUN,
HUA SUMING,
TOYOSHIMA CHIKASHI
Publication year - 2003
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1111/j.1749-6632.2003.tb07140.x
Subject(s) - chemistry , binding site , cleavage (geology) , enzyme , substrate (aquarium) , mutant , atpase , nucleotide , stereochemistry , binding domain , biochemistry , biology , paleontology , ecology , fracture (geology) , gene
A bstract : The role of amino acid residues involved in substrate and cation binding was investigated in complementary experiments on Fe 2+ ‐catalyzed oxidation and cleavage, limited digestion with proteinase K, and mutational analysis. Cleavage at Ser346 was produced by Fe 2+ in the presence of substrate (ATP or AMP‐PNP) and Ca 2+ , and was attributed to Fe 2+ bound to a Mg 2+ site near Ser346 and neighboring Glu696. Ca 2+ ‐ and ATP‐dependent oxidation of the Thr441 side chain was also observed and attributed to Fe 2+ substituting for Mg 2+ in the Mg 2+ ‐ATP complex bound to the N domain. Mutation of Arg560 or Glu439 within the N domain interfered with nucleotide‐dependent ATPase resistance to digestion with proteinase K. Furthermore, mutation of Lys352, Lys684, Thr353, Asp703, or Asp707 within the P domain produced similar interference, consistent with a role of these residues in substrate stabilization at the catalytic site. In a third group of experiments, equilibrium isotherms were obtained with Asn796Ala and Glu309Gln mutants, demonstrating non‐cooperative binding of one Ca 2+ per ATPase, as opposed to cooperative binding of two Ca 2+ by WT enzyme. No high‐affinity binding by Asp800Asn, Glu771Gln, and Thr799Ala mutants was detected. It was also demonstrated that the conformational transitions involved in enzyme activation and interconversion of Ca 2+ binding and phosphorylation energy, are triggered by Ca 2+ binding to site II and stabilization of Glu309 (M4) and N796 (M6).